Therapeutic somatostatin analogs

ABSTRACT

An octapeptide of the formula: ##STR1## wherein each A 1  and A 2 , independently, is H, C 1-12  alkyl, C 7-10  phenylalkyl, R 1  CO (where R 1  is C 1-20  alkyl, C 3-20  alkenyl, C 3-20  alkinyl, phenyl, naphthyl, or C 7-10  phenylalkyl), or R 2  OCO (where R 2  is C 1-10  alkyl or C 7-10  phenylalkyl), provided that when one of A 1  or A 2  is R 1  CO or R 2  OCO, the other must be H; A 3  is CH 2  -A 6  (where A 6  is pentafluorophenyl, naphthyl, pyridyl, or phenyl); A 4  is o- m- or p-substituted X-Phe (where X is a halogen, H, NO 2 , OH, NH 2 , or C 1-3  alkyl), pentafluoro-Phe, or β-Nal; A 5  is Thr, Ser, Phe, Val, α-aminobutyric acid, or Ile, provided that when A 3  is phenyl, A 1  is H, and A 2  is H, A 5  cannot be Val; and A 7  is Thr, Trp, or β-Nal; or a pharmaceutically acceptable salt thereof.

BACKGROUND OF THE INVENTION

This application is a continuation of co-pending application Ser. No.209,883 filed on June 22, 1988, which is a continuation in part of Coyet al. U.S. Ser. No. 070,400, filed July 7, 1987, which is acontinuation in part of Coy et al. U.S. Ser. No. 010,349, filed Feb. 3,1987, which is a continuation in part of Coy et al. U.S. Ser. No.875,266, filed June 17, 1986, which is a continuation in part of Coy etal. U.S. Ser. No. 775,488, filed Sept. 12, 1985.

This invention relates to therapeutic peptides.

A number of somatostatin analogs exhibiting GH-release-inhibitingactivity have been described in the literature, including analogscontaining fewer than the naturally occurring fourteen amino acids. Forexample, Coy et al. U.S. Pat. No. 4,485,101, hereby incorporated byreference, describes dodecapeptides having an N-terminal acetyl group, aC-terminal NH₂, D-Trp at position 6, and p-Cl-Phe at position 4.(Herein, when no designation of configuration is given, the L-isomer isintended )

SUMMARY OF THE INVENTION

In general, the invention features an octapeptide of the formula:##STR2## wherein each A₁ and A₂, independently, is H, C₁₋₁₂ alkyl, C₇₋₁₀phenylalkyl, R₁ CO (where R₁ is C₁₋₂₀ alkyl, C₃₋₂₀ alkenyl, C₃₋₂₀alkinyl, phenyl, naphthyl, or C₇₋₁₀ phenylalkyl), or R₂ OCO (where R₂ isC₁₋₁₀ alkyl or C₇₋₁₀ phenylalkyl), provided that when one of A₁ or A₂ isR₁ CO or R₂ OCO, the other must be H; A₃ is CH₂ --A₆ (where A₆ ispentafluorophenyl, naphthyl, pyridyl, phenyl, or o-, m-, or, morepreferably, p-substituted phenyl, where the substituent is a halogen,NH₂, NO₂, OH, or C₁₋₃ alkyl); A₄ is o-, m- , or, more preferably,p-substituted X-Phe (where X is a halogen, H, NH₂, NO₂, OH, or C₁₋₃alkyl), pentafluoro-Phe, or β-Nal; A₅ is Thr, Ser, Phe, Val,α-aminobutyric acid, or Ile, provided that when A₃ is phenyl, A₁ is H,and A₂ is H, A₅ cannot be Val; and A₇ is Thr, Trp, or β-Nal; or apharmaceutically acceptable salt thereof.

In the formula given above, the configuration of the molecule at thecarbon atom to which A₃ is bonded is not given, to indicate that theamino acid residue of which A₃ is a substituent can have the D- or L-configuration. In the formula given above, there is a bond shown betweenthe two Cys residues to indicate cyclization; in all of the compounds ofthe invention there is such cyclization, but the Cys-Cys bond lines areomitted for convenience.

Preferred compounds of the invention includeD-β-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH₂ ;D-Phe-Cys-Tyr-D-Trp-Lys-α-Aminobutyric acid-Cys-Thr-NH₂ ;pentafluoro-D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH₂ ;N-Ac-D-β-Nal-Cys-Tyr-D Trp-Lys-Val-Cys-Thr-NH₂ ;D-β-Nal-Cys-pentafluoro-Phe D-Trp-Lys-Val-Cys-Thr-NH₂ ;D-β-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-β-Nal-NH₂ ;D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-β-Nal-NH₂ ;D-β-Nal-Cys-Tyr-D-Trp-Lys-α-aminobutyric acid-Cys-Thr-NH₂ ;D-p-Cl-Phe-Cys-Tyr-D-Trp-Lys-α-aminobutyric acid-Cys-Thr-NH₂ ; andacetyl-D-p-Cl-Phe-Cys-Tyr-D-Trp Lys-α-aminobutyric acid-Cys-Thr-NH₂ ;and D-Phe-Cys-β-Nal-D-Trp-Lys-Val-Cys-Thr-NH₂.

In other preferred embodiments, a therapeutically effective amount ofthe therapeutic compound and a pharmaceutically acceptable carriersubstance (e.g. magnesium carbonate, lactose, or a phospholipid withwhich the therapeutic compound can form a micelle). The most preferredcarrier substance is mannitol. Examples of such compositions include apill, tablet, capsule, or liquid for oral administration to a humanpatient, a spreadable cream, gel, lotion, or ointment for application tothe skin of a human patient in need of the compound, a liquid capable ofbeing administered nasally as drops or spray, or a liquid capable ofintravenous, parenteral, subcutaneous, or intraperitonealadministration. The pill, tablet or capsule can be coated with asubstance capable of protecting the composition from the gastric acid inthe patient's stomach for a period of time sufficient to allow thecomposition to pass undisintegrated into the patient's small intestine.The therapeutic composition can also be administered in the form of anoil emulsion or dispersion in conjunction with a lipophillic salt suchas a pamoic acid. The therapeutic composition can also be in the form ofa biodegradable sustained release formulation for intramuscularadministration. For maximum efficacy, zero order release is desired.Zero order release can be obtained using an implantable or externalpump, e.g., Infusaid ™ pump, to administer the therapeutic composition.

The compounds of the invention are active in inhibiting the secretion ofGH, insulin, and glucagon. Further, the aromatic lipophilic N-terminalend can provide long-lasting in vivo activity.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Structure

The compounds of the invention have the general formula recited in theSummary of the Invention, above. They are all octapeptide analogs ofsomatostatin which have D-Trp at position 4; and optional modificationsat positions 3 (A₄) 6 (A₅) and 8 (A₇). It has been found thatD-β-naphthylalanine at positions 1 and/or 3; Tyr at position 3; and Valat position 6 are modifications which particularly enhance activity.

The compounds can be provided in the form of pharmaceutically acceptablesalts or complexes. Examples of preferred salts or complexes are thosewith therapeutically acceptable organic acids, e.g., acetic, lactic,maleic, citric, malic, ascorbic, succinic, benzoic, salicylic,methanesulfonic, toluenesulfonic, or pamoic acid, as well as polymericacids such as tannic acid or carboxymethyl cellulose, and salts withinorganic acids such as the hydrohalic acids, e.g., hydrochloric acid,sulfuric acid, or phosphoric acid.

Synthesis

The synthesis of one octapeptide follows. Other octapeptides of theinvention can be prepared by making appropriate modifications, withinthe ability of someone of ordinary skill in this field, of the followingsynthetic method.

The first step in the preparation ofD-β-naphthylalanine-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr- NH₂ was thepreparation of the intermediatetert-butyloxycarbonyl-D-β-naphthylalanine-S-methylbenzyl-Cys-Tyr-D-Trp-N.sup.ε-benzyloxycarbonyl-Lys-Val-S-methylbenzyl-Cys-O-benzyl-Thr-benzyhydrylamineresin, as follows.

Benzhydrylamine-polystyrene resin (Vega Biochemicals, Inc.) in thechloride ion form was placed in the reaction vessel of a Beckman 990Bpeptide synthesizer programmed to perform the following reaction cycle:(a) methylene chloride; (b) 33% trifluoroacetic acid in methylenechloride (2 times for 1 and 25 min each); (c) methylene chloride; (d)ethanol; (e) methylene chloride; (f) 10% triethylamine in chloroform.

The neutralized resin was stirred with Boc-O-benzyl-threonine anddiisopropylcarbodiimide (1.5 mmole each) in methylene chloride for 1 hand the resulting amino acid resin was then cycled through steps (a) to(9) in the above wash program. The following amino acids (1.5 mmole)were then coupled successively by the same procedure:Boc-S-methylbenzyl-Cys, Boc-Val, Boc-Ne-benzyloxycarbonyl-lysine,Boc-D-Trp, Boc-Tyr, Boc-S-methylbenzyl-Cys, Boc-D-β-naphthylalanine.

The resin was washed and dried and then mixed with anisole (4 ml) andanhydrous hydrogen fluoride (36 ml) at 0° C. and stirred for 45 min.(one can also use thioanisole, trifluoroacetic acid, andtrifluoromethane sulfonic acid at a ratio of 1:90:9, for 6 h). Excesshydrogen fluoride was evaporated rapidly under a stream of dry nitrogenand free peptide precipitated and washed with ether. The crude peptidewas then dissolved in 800 ml of 90% acetic acid to which was added I₂ inmethanol until a permanent brown color was present. The solution wasthen stirred for 1 h before removing the solvent in vacuo. The resultingoil was dissolved in a minimum volume of 50% acetic acid and eluted on acolumn (2.5×100 mm) of Sephadex G-25. Fractions containing a majorcomponent by uv absorption and thin layer chromatography were thenpooled, evaporated to a small volume, and applied to a column (2.5×50cm) of Whatman LRP-1 octadecylsilane (15-20 μ M).

The column was eluted with a linear gradient of 10-50% acetonitrile in0.1% trifluoroacetic acid in water. Fractions were examined by thinlayer chromatography and analytical high performance liquidchromatography and pooled to give maximum purity and if desired, adifferent salt prepared, e.g., acetate or phosphate. Repeatedlyophilization of the solution from water gave 170 mg of the product asa white, fluffy powder.

The product was found to be homogeneous by Hplc and Tlc. Amino acidanalysis of an acid hydrolysate confirmed the composition of theoctapeptide.

The octapeptides of the invention having the formulaepentafluoro-D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH₂, D-Phe-Cys-Tyr-D-TrpLys-α-aminobutyric acid-Cys-Thr-NH₂,N-Ac-D-β-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH₂, D-β-Nal-Cyspentafluoro-Phe-D-Trp-Lys-Val-Cys-Thr-NH₂, D-β-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-β-Nal-NH₂, D-Phe-Cys-Tyr-D-Trp Lys-Val-Cys-β-Nal-NH₂ ;D-β-Nal-Cys-Tyr-D-Trp-Lys-α-aminobutyric acid-Cys-Thr-NH₂ ;D-p-Cl-Phe-Cys-Tyr-D-Trp-Lys-α-aminobutyric acid-Cys-Thr-NH₂ ;acetyl-D-p-Cl-Phe-Cys-Tyr-D-Trp-Lys-α-aminobutyric acid-Cys-Thr-NH₂ ;and D-Phe-Cys-β-Nal-D-Trp-Lys-Val-Cys-Thr-NH₂ were made according tomethods analogous to those described above.

Use

When administered to mammals, particularly humans, (e.g. orally,topically, intravenously, parenterally in a sustained release,biodegradable form, nasally, or by suppository), the compounds can beeffective to inhibit GH release as well as to inhibit insulin, glucagon,and pancreatic exocrine secretion, and to therapeutically affect thecentral nervous system.

The compounds can be administered to a mammal, e.g. a human, in thedosages used for somatostatin or, because of their greater potency, insmaller dosages. The compounds of the invention can be used for thetreatment of cancer, particularly growth hormone-dependent cancer (e.g.,bone, cartilage, pancreas (endocrine and exocrine), prostate, orbreast), acromegaly and related hypersecretroy endocrine states, or ofbleeding ulcers in emergency patients and in those suffering frompancreatitis or diarrhea. The compounds can also be used in themanagement of diabetes and to protect the liver of patients sufferingfrom cirrhosis or hepatitis. The compounds can also be used to treatAlzheimer s disease, as analgesics to treat pain by acting specificallyon certain opiate receptors, and as gastric cytoprotective compounds forulcer therapy. The compounds can also be used to treat certain types ofmushroom poisoning.

The compounds can also be used to treat diabetes-related retinopathy.The anti-cancer activity of the compounds may be related to theirability to antagonize cancer-related growth factors such as epidermalgrowth factor.

The compounds can be administered to a mammal, e.g., a human, in adosage of 0.01 to 50 mg/kg/day, preferably 0.1 to 5 mg/kg/day.

Other embodiments are within the following claims.

We claim:
 1. An octapeptide of the formula: ##STR3## wherein X is H, OH,CH₃, or a halogen, or a pharmaceutically acceptable salt thereof.
 2. Theoctapeptide of claim 1 wherein X is H.
 3. A therapeutic compositioncapable of inhibiting the release of growth hormone, insulin, glucagon,or pancreatic exocrine secretion comprising a therapeutically effectiveamount of the compound of claim 1 together with a pharmaceuticallyacceptable carrier substance.
 4. A method of treating a mammal in needof reduction of growth hormone, insulin, glucagon, or pancreaticexocrine secretion comprising administering to said mammal atherapeutically effective amount of the compound of claim
 1. 5. Thetherapeutic composition of claim 3 wherein said composition is in theform of a pill, tablet, or capsule for oral administration to a humanpatient in need of said compound.
 6. The therapeutic composition ofclaim 3 wherein said composition is in the form of a liquid for oraladministration to a human patient in need of said compound.
 7. Thetherapeutic composition of claim 5, said composition being coated with asubstance capable of protecting said composition from the gastric acidin the stomach of said human patient for a period of time sufficient toallow said composition to pass undisintegrated into the small intestineof said human patient.
 8. The therapeutic composition of claim 3, saidcomposition being in the form of a cream, gel, spray, or ointment forapplication to the skin of a human patient in need of said compound. 9.The therapeutic composition of claim 3, said composition being in theform of a liquid capable of being administered nasally as drops or sprayto a human patient in need of said compound.
 10. The therapeuticcomposition of claim 3, said composition being in the form of a liquidfor intravenous, subcutaneous, parenteral, or intraperitionealadministration to a human patient in need of said compound.
 11. Thetherapeutic composition of claim 3, said composition being in the formof a biodegradable sustained release composititon for intramuscularadministration to a human patient in need of said compound.